This grant has as its goal the elucidation of the mechanisms by which the phosphatidylinositol signalling system evokes intracellular responses to extracellular agonists. Understanding this system will provide new insights into platelet physiology and pathology and also into the proliferation and differentiation of megakaryocytes. The experiements will also address the pathogenesis of several disease states in which defects in inositol signalling are present. In particular, the applicant will study the relationship between inositol polyphosphate 4-phosphatase (4-Ptase) and phosphatidylinositol 3-kinase in platelets. He will investigate the mechanism by which 4-Ptase controls megakaryocyte proliferation in GATA-1 null megakaryocytes. In the absence of 4-Ptase, these megakaryocytes proliferate continuously and fail to produce platelets. Restoration of 4-Ptase arrests megakaryocyte growth. He will use NIH 3T3 cells to investigate whether this growth arresting property of 4-Ptase is general. He will investigate the homolog of 4-Ptase, SopB, a Salmonella gene required for virulence. In the absence of SopB, the organisms infect intestinal epithelia but fail to induce neutrophilic infiltration and diarrhea. SopB is an inositol phosphatase and enzyme activity is required for virulence. The substrate specificity and derangement of inositol metabolism in infected cells will be determined. He will study another homolog of 4-Ptase that is a tumor suppressor gene (PTEN) and is also an inositol phosphatase. The OCRL-1 5-phosphatase that when mutated is the cause of Lowe Syndrome will be examined. The applicant propose that the defect results in abnormal targeting of lysosomal enzyme in Lowe Syndrome. He plans to study this and to measure plasma lysosomal enzymes in patients with Lowe Syndrome. He will also attempt to elucidate the enzymology and regulation of production of isomers of inositol tetraphosphates and inositol pentaphosphates. He will identify, isolate, and clone cDNA for enzymes leading to InsP5 starting with inositol 1,3,4-triphosphate 5/6-kinase.